1. Field of the Invention
This invention relates to a method for the preparation of purified antibodies which are specific for native homologous hapten or antigen. The invention also relates to novel artificial antigens useful for generating such specific antibodies. The antibodies produced in accordance with this invention are useful in immune assay of a wide variety of chemical substances and in particular those of pharmacologic and/or social significance such as the steroids, catechol amines, prostaglandins and drugs of abuse.
This application is a continuation in part of my prior, copending applications Ser. Nos. 45,558, filed June 11, 1970, and 89,929, filed Nov. 16, 1970. The immunologic terms employed herein are believed to be in accord with conventional usage and definition. Should any presently unforseen confusion arise, unless otherwise indicated, the construction of a term shall be in accordance with its definition and usage in the well-known textbook by Weiser, Myrvid and Pearsall, Fundamentals of Immunology for Students of Medicine and Related Sciences, published by Lea & Febiger, Philadelphia, 1969.
2. Description of the Prior Art
A relatively new approach to biological assaying involves immunochemical procedures as a basis for the assay. Such procedures involve the use of antibodies which react with the compound to be assayed. A known amount of antibody and the sample, obtained from the test species, are intermixed. Theoretically, if the antibody is specific for the compound to be assayed (i.e., does not cross react with biologically distinct structural homologs or analogs) one could then accurately measure the amount of antibody reacting with the test compound using conventional radioimmune or fluorescent competition assay techniques. This amount can then be translated into the amount of test compound present.
To date no immunochemical technique has been developed which produces a reliable, reproducibly accurate assay employing such immunochemical procedures. The problem, aside from the lack of sensitivity of available measurement techniques, has been that the antibodies produced in accordance with presently available methods are not specific for the test compound. These prior art antibodies cross-react with the biologically distinct analogs or homologs of the test compound encountered in the serum sample under assay.
Antibodies to immunogenic compounds of high molecular weight, such as proteins, can be produced by administering the unaltered or natural compound to the antibody producing host. However, small molecules which are not immunogenic by themselves, e.g. steroids, drugs of abuse, prostaglandins or catechol amines, must be bound to a high molecular weight immunogenic carrier. Furthermore, it has, in certain cases, been found desirable to couple antigenic compounds to carrier molecules, for example, in attempts to reproduce high yields of antibody to such antigens. Such artificial antigens induce antibody formation. Substances which do not induce the formation of antibody, unless bound to a high molecular weight carrier, are herein termed "haptens" in conformance with conventional usage.
Conventionally artificial antigens have been produced by conjugating immunogenic carrier molecules through the reactive functional groups on the haptenic molecule, e.g. see J. Biol. Chem., 228:713 (1957); id, 234:1090 (1958); Can. J. Biochem. Physiol., 36:1177 (1958); id., 39:941, 961 (1961); Science, 129:564 (1959); J. Immun., 92:515 (1964); Biochem. 9:331 (1970); Science, 168:1347 (1970) and J. Pharmacol. 178:253 (1971). Such attempts to produce specific antibodies by coupling carrier molecules to one of the functional groups of the hapten have successfully rendered the hapten immunogenic. However, considerable cross reactivity is demonstrated by biologically distinct structural analogs coupled at the same or similar sites. Such non-specific binding with related compounds negates the specificity of these conventionally produced prior art antibodies. See Steroids, 16:387 (1970); id 18:555, 593, 605 (1971); Karolinska Symposia on Research Methods In Reproductive Endocrinology, p. 320, Ed. E. Diczfalusky Bogtrykksiut Forum, Copenhagen (1970) and "Immunologic Methods in Steroid Determination", Eds. Peron and Caldwell, Appleton-Century-Crofts, New York (1970), p. 41.
Another approach to the preparation of specific antibodies to small molecules involved coupling the immunogenic carrier directly into the aromatic ring of the hapten. For example, see Immunochemistry 5:55 (1968) describing the synthesis of certain immunogenic steroid-protein conjugates and the production of rabbit antiserum to beta-estradiol, coupled to bovine serum ilbunin (BSA) and keyhole limpet hemocyanin (KLH). Antisera were tested against steroids coupled to human gamma globulin (IgG). The immunological assays employed quantitative precipitin and hapten inhibition tests. The conjugates produced in accordance with the method described in this paper did not produce antibodies which were specific for the steriod hapten (i.e. beta-estradiol, estriol and estrone) employed in the conjugate. Antibody to estradiol-KLH brought down a non-specific precipitate with testosterone-IgG, and antibody to estriol-KLH brought down a precipitate (nonspecifically) with testosterone-IgG and with estradiol-IgG. For a similar approach, see also Chem. Abstracts, 39:1505 (1945) in which azo proteins of morphine and strychnine were alleged to have been prepared by coupling to diazotized acetyl-p-phenylenediamine. Antibodies generated with the immunogens prepared in accordance with this paper also exhibit undesirable cross reactivity.
Most recently an attempt to produce specific antibodies for 17-beta-estradiol by coupling bovine serum albumin via the alicylic C.sub.6 position was reported in Steroids, 18:605 (1971). The reported results do not support the claimed specificity.
Therefore, to the best of my knowledge, the art is devoid of a method, employing artificial antigens for preparation of purified antibodies which are indeed truly specific; that is, antibodies which do not cross react to any significant extent with biologically distinct, structural analogs or homologs of the native homologous hapten or antigen.
To understand the importance of specificity to practical immunochemical competition assays, one need only consider that morphine specific antibodies are clearly mandatory for development of a commercially practical morphine assay. Such an assay must distinguish morphine from codeine, the commonly dispensed 3-methoxy morphine, and for that matter, from the important synthetic surrogates of morphine (e.g. methadone, meperidine and pentazocine.) The same is true, for example, in the estrogen series where cross reactivity within the class (i.e. estrone, estradiol and estriol) will defeat the usefulness of the assay.
The provision of purified antibodies exhibiting such specificity is a primary object of this invention. The antibodies produced in accordance with this invention do not cross react to any significant degree with biologically distinct structural homologs or analogs of their homologous haptens or antigens.